In all tissues of the mammalian body, there is a similar response of wounded cellular elements to injury. Injury is a process which is induced by either trauma (physical, chemical, thermal, electrical), or which results from diseases which produce acute or chronic inflammation. A fundamental phenomenon of wound healing is the metabolic activation of fibroblast cells. These cells produce the protein collagen which comprises the major portion of scar tissue.
The following brief discussion concerning the biochemistry of collagen synthesis serves to define the manner in which the wound healing response can result in permanent structural damage.
Basically, collagen molecules are synthesized within the endoplasmic reticulum of fibroblasts, stored within the Golgi apparatus, and from there are extruded into the extracellular space as procollagen. Outside of the cell, the procollagen molecules spontaneously arrange themselves by non-chemical forces into fibrils called tropocollagen. In this type of collagen, there are no chemical cross-links between individual molecules, despite a morphological appearance of mature collagen. The absence of chemical cross-linking causes the collagen to remain in an immature state. Immature collagen is characterized by a very low tensile strength, hence the scar tissue is present in usual amounts but is weak and easily pulled apart. Immature collagen is further characterized by its solubility in saline. The maturation of collagen fibrils is directly related to the formation of cross-links between individual collagen molecules.
The cross-linking of collagen molecules is controlled by the enzyme lysyl oxidase. The cross-linking occurs in two stages with the first stage involving the enzymatic synthesis of aldehydes in the presence of lysyl oxidase. This stage is shown by the following chemical reaction: ##STR1##
The reaction involves the removal of terminal amino groups from lysyl or hydroxylysyl residues in the tropocollagen. The copper-containing enzyme lysyl oxidase serves as the catalyst. After the first stage is complete and the aldehydes are produced, one of the two following cross-linking reactions may occur: ##STR2##
Reaction A produces a stable and irreversible intermolecular bond, with no further reactions necessary.
Reaction B involves the reversible production of a Schiff base, which is subsequently reduced to produce a stable cross-link. Both of the cross-linking reactions involve intermolecular bonding which significantly increases the tensile strength of collagen and thereby results in a mature collagen fiber.
It is known that the cross-linking of collagen fibrils can be prevented by the use of agents which have their effect either by inhibiting the enzyme, lysyl oxidase, or by binding to the aldehydes produced as a result of its action. Beta-aminopropionitrile (BAPN), aminoacetonitrile, beta-mercaptoethylamine, dithiothreitol, isoniazide, iproniazide, carbonyl reagents, disulfhydryls and diamines are all known to cause in vitro inhibition of lysyl oxidase.
D-pencillamine is known to have two effects on the collagen cross-linking process: to reversibly inhibit the lysyl oxidase; and to irreversibly bind to the aldehydes generated as a result of its action. BAPN and d-pencillamine apparently have the best in vivo effects of the above mentioned agents, and, therefore, have been the most closely investigated.
BAPN was first isolated from sweet peas, or Lathyrus odoratus. Interestingly, it was farmers whose cattle grazed on sweet peas who noted that their cattle developed bone and joint abnormalities and that some died from ruptured aortic aneurysms. It was discovered that this condition, now known as lathyrism, resulted from BAPN's inhibition of lysyl oxidase.
The inhibition of lysyl oxidase has been disclosed in the literature. Page and Benditt postulated that the mechanism of BAPN-induced lathyrism is due to its irreversible inhibition of lysyl oxidase in 1967 in Biochemistry, Vol. 6, pages 1142-1147 and in the Proceedings of the Society for Experimental Biology and Medicine, Vol. 124, pages 454-459.
BAPN has been used systemically in humans to control the tensile strength of fibrous tissue involved in scleroderma, flexor tendon repair, and urethral stricture. Keiser and Sjoerdsma attempted to treat humans afflicted wich scleroderma with orally-administered BAPN and disclosed their results in Clinical Pharmacology and Therapeutics, Vol. 8, No. 4, pps. 593-602. Four patients were treated for 22 to 67 days. A reversible periosteal reaction occurred in one patient and untoward effects were observed in patients at a dose level which produced only moderate effects on skin collagen and no apparent therapeutic effect. The authors proposed that future use of the drug as an inhibitor of collagen cross-linking in man should be limited to short term trials.
In a report entitled, "Some studies on the effects of Beta-aminopropionitrile in patients with injured flexor tendons," published in 1969 in Surgery, Vol. 66, pps. 215-223, Peacock and Madden disclosed that they orally administered BAPN to humans following repair of flexor tendons but discontinued the study because of undesirable systemic reaction. Another publication by Peacock et al. entitled, "Administration of Beta-aminopropionitrile to Human Beings with Urethreal Strictures: A Preliminary Report," published in November, 1978, Vol. 136 issue of the American Journal of Surgery, reported the reduction in breaking strength of newly formed connective tissue in patients systemically treated with very low doses of BAPN. The patients did not develop signs of toxicity; however, the clinical proof of their improvement as a result of the BAPN systematic treatment was equivocal.
Apparently, the many side effects associated with the systemic use of BAPN have prevented the widespread acceptance of the inhibitor by the medical profession. A February 1972 article entitled, "Caution Against the Use of Lathyrogens," by Barrow et al. appearing on pages 309-310 in Vol. 71, No. 2 of Surgery, discloses BAPN's function as a lathyrogenic agent but urges great caution in the use of such agents.
Other publications have disclosed the effects of other compositions known to inhibit collagen fibril cross-linking. One such disclosure is made in ARVO abstracts, 1980 by Brancato et al. in which it is reported that the oral administration of d-penicillamine to rabbits for 14 days resulted in an increase in the solubility of vitreous collagen. No clinical proof of the efficacy of oral administration of d-penicillamine was reported. A 1976 publication entitled, "Lathyritic Activity of Isoniazid," appearing in Vol. 7, Nos. 3 and 4 of the Journal of Medicine suggests that the therapeutic effectiveness of isoniazide as an antituberculous drug might reside in its lathyritic activity.
One of the difficulties with compositions which are utilized to inhibit collagen cross-linking is that some of the composition may be metabolized before its lathyritic action is initiated or completed. It is desirable to limit the metabolism of collagen cross-linking inhibitors under certain circumstances. A 1979 article entitled, "Effect of Pargylene on the Metabolism of BAPN by Rabbits," Toxicology and Applied Pharmacology 47, pps. 61-69 discloses the reduction of BAPN degradation by the monoamine oxidase inhibitor pargylene and suggests that the chemical might be useful in prolonging the biological activity of BAPN.
Although the literature discloses the systemic use of collagen cross-linking inhibitors in the treatment of either trauma-induced or disease-induced wounds, it would appear that such a use has not received widespread medical acceptance.
Scarring in tissue located anywhere in the body may cause permanent alterations in the usual anatomical structures of the wounded tissue. Nowhere are complications from scarring more pronounced than in ocular tissue. Contracting collagen fibers produce traction which can cause very undesirable conditions in ocular tissue. After trauma to the external ocular structures, for example, undesirable effects may result from malrotation of the lids, trichiasis, and fornix contracture. Diseases which are external to the eyeballs can also cause difficulties. In cicatricial inflammatory diseases such as pemphigoid and Stevens-Johnson syndrome, the fornix of the eye may shrink and be completely obliterated.
There are other situations in which contraction caused by the maturation of collagen in healing ocular wounds can result in undesirable effects. In the surgical procedure to correct near-sightedness, radial keratotomy, the cornea is subjected to numerous deeply penetrating incisions. The results of this surgery are quite variable It is likely that the unpredictability of the surgical results is associated with the contraction of collagen fibers during the healing of the incisions.
The problems attendant to cataract surgery present another area in which contracting collagen fibers can cause difficulties. Anterior chamber intraocular lenses may be dislocated by contracting peripheral anterior senechiae and the pupil may be distorted as a result of scarring.
There are still other difficulties which scarring of ocular tissue poses to the patient as well as the ophthalmologist. Retinal traction and sometimes detachment may be caused by vitreous collagen contraction and shrinking. Surgical procedures utilized in the treatment of glaucoma many times are unsuccessful. For example, the failure of glaucoma filtering procedures may be caused by the contraction and tightening of cicatricial collagenous bands stretching across the fistula.
Most of the difficulties which the ophthalmologist faces by virtue of contracting collagen fibers have been dealt with surgically. It would be desirable to reduce or completely eliminate the undesirable effects caused by the contraction of maturing collagen fibers located in ocular tissue by the employment of a safe, relatively inexpensive and expedient method.